Despite overwhelming evidence that overexpression of apoA-I inhibits progression and even induces regression of atherosclerosis in animals, there remain substantial questions about the mechanisms by which apoA-I reduces atherosclerosis as well as specific questions about the structure-function properties of apoA-I with regard to its ability to promote reverse cholesterol transport (RCT) and reduce atherosclerosis. Project 3 will focus on the use of in vivo studies in mice to address some of these questions in a manner that is highly interactive with Projects 1 and 2. One of the major questions to be addressed is whether intervention to increase plasma apoA-I does in fact promote RCT, a topic of continued debate and uncertainty. We will use different approaches for quantitating RCT in mice. Quantitation of fecal sterol excretion will be used as a measure of total peripheral RCT. In order to determine the rate of RCT specifically from the macrophage, a novel tracer method in which macrophage foam cells labeled with 3H-cholesterol are injected and labeled cholesterol and bile acids are traced in the feces. We will also use a variety of other measures including serum efflux capacity (in collaboration with Project 1), HDL subclass distribution, LCAT activation, and assessment of atherosclerosis. In Specific Aim 1, we will ask whether overexpression of human apoA-I promotes macrophage RCT and whether this correlates with reduction in atherosclerosis. In Specific Aim 2, the importance of the apoA-I interaction with other key gene products in the RCT pathway, such as ABCA1, LCAT, and SR-BI, will be tested using in vivo mouse models. In Specific Aim 3, similar methods will be used to express mutants of apoA-I (in collaboration with Project 2) to probe the structure-function relationships of apoA-I in vivo with regard to effects on RCT and atherosclerosis. Several of the mutant and chimeric forms of apoA-I that will be studied intensively in vitro for lipid binding properties in Project 2 will be assessed in vivo in Project 3. In summary, Project 3 represents in vivo studies that directly address the role of apoA-I in promoting RCT and the importance of interaction with other genes and of the structure of apoA-I in determining its effects. This project complements the work being performed in Projects 1 and 2 and will result in a highly collaborative effort among the 3 projects in this Program Project.